The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. ‘LOV-Turbo’ works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.
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The data associated with this study are available in the article and the Supplementary Information. The original mass spectra, spectral library and the protein sequence database used for searches have been deposited in the public proteomics repository MassIVE (http://massive.ucsd.edu) and are accessible at ftp://massive.ucsd.edu/MSV000090683/. Additional data beyond that provided in the figures and Supplementary Information are available from the corresponding author on request.
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Rat cortical neurons were a kind gift from M. Lin (Stanford University). We thank J. Reinstein (Max Planck Institute) for helpful feedback. This work was supported by the NIH (grant nos. R01-DK121409 and RC2DK129964 to A.Y.T., R01-OD026223 to A.Y.T. and S.A.C. and T32GM007276 to J.S.C.), the Stanford Wu Tsai Neurosciences Institute (A.Y.T.), the National Science Foundation (NeuroNex grant no. 2014862 to A.Y.T. and GRFP DGE-1656518 to J.S.C.), the National Research Foundation of Korea grant no. NRF-2019R1A6A3A03033677 (S.-Y.L.), the Stanford Gerald J. Lieberman Fellowship (J.S.C.) and the Burroughs Wellcome Fund grant no. CASI 1019469 (C.K.K.). A.Y.T. is a Chan Zuckerberg Biohub – San Francisco Investigator.
S.-Y.L., J.S.C. and A.Y.T. have filed a patent application covering some aspects of this work (US Provisional Patent Application No. 63/488,940; CZ SF ref. CZB-273S-P1; Stanford ref. S22-487; KT ref. 110221-1361830-009500PR). The remaining authors declare no competing interests.
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Supplementary Figs. 1–8, Table 1, Legends of Tables 2–4, Note 1, Methods, Antibodies list, Genetic constructs list and References.
Supplementary Table 2–4
Table 2: Proteomic data for ERM to nucleus pulse-chase experiment. Table 3: Proteomic data for ERM to mitochondria pulse-chase experiment. Table 4: Proteomic data at peptide level for ERM to nucleus pulse-chase experiment.
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Lee, SY., Cheah, J.S., Zhao, B. et al. Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells. Nat Methods 20, 908–917 (2023). https://doi.org/10.1038/s41592-023-01880-5